Immunomodulation using altered dendritic cells

ABSTRACT

The invention relates to altered immune cells and their use in methods and compositions to alter the immune system in a mammal. More specifically, the invention is directed to the alteration of gene expression in antigen presenting cells such as dendritic cells (DC) and their use in various methods and compositions to alter T cell activity for the treatment of a variety of immune disorders.

FIELD OF THE INVENTION

The invention relates to altered immune cells and their use in methodsto alter the immune system in a mammal. More specifically, the inventionis directed to the alteration of gene expression in dendritic cells (DC)and their use in various methods to alter T cell activity for thetreatment of a variety of immune disorders.

BACKGROUND OF THE INVENTION

Throughout this application, various references are cited in parenthesesto describe more fully the state of the art to which this inventionpertains. Full bibliographic information for each citation is found atthe end of the specification, immediately preceding the claims. Thedisclosure of these references are hereby incorporated by reference intothe present disclosure.

Dendritic cells (DC) are the most potent antigen presenting cell (APC)endowed with the unique ability to stimulate and polarize naïve T cellsto either Th1 or Th2 phenotypes (Maldonado-Lopez, R. et al., 2001.13:275). DC also play a critical role in the maintenance of selftolerance by curtailing T cell responses directly or indirectly throughthe generation of T regulatory cells (Belz, G. T., et al., 2002. lmmunolCell Biol 80:463; Mahnke, K., et al., 2002. Immunol Cell Biol 80:477;Min W. P. et al., J. Immunol. in press). The difference between DCsubsets that stimulate and those that suppress immune responses seems toreside in the expression of co-stimulatory molecules and cytokines(Jonuleit, H., et al., 2001. Trends Immunol 22:394; Lu, L., et al.,2002. Transplantation 73:S19). The subset of DC called tolerogenic DC(Tol-DC) have a distinct phenotype, suppress activation of conventionalT cells and activate T regulatory cells (Treg) in an antigen-specificmanner (Chang, C. C. et al., 2002. Nat Immunol. Mar.; 3(3), 237-43;Gilliet M., et al.,2002. J. Exp. Med. March 18;195(6):695-704; Roncarlo,M. G. et al., 2001. J. Exp. Med. January 15;193(2):F5-9; Kawahata, K.,et al., 2002. February 1; 168(3):1103-12.). Tol-DC possess reducedexpression of the co-stimulatory molecules CD40, CD80 and CD86 andreduced ability to secrete T cell activating cytokines such asinterleukin-12. Generally, expression of interleukin-12 (IL-12) seems tostimulate Th1 activation (O'Garra, A., et al., 1995. Res Immunol.146:466), whereas production of IL-10 by DC stimulates Th2 activation(Liu, L., et al., 1998. Int Immunol 10:1017), and in some casesregulatory T cell generation (Akbari, O., et al., 2001. Nat Immunol2:725; McGuirk, P., et al., 2002. J. Exp Med 195:221). Understandingthis duality in function has led to DC based immunotherapies, which havebeen used to potentiate T cell responses (in the case of cancervaccines) or diminish them (in autoimmune disorders and transplantation)(Pardoll, D. M. 1998. Cancer Vaccines. Nat Med 4:525; Morel, P. A. etal., 2001. Trends Immunol. 22:546; Prud'homme, G. J. 2000. J Gene Med2:222).

Tolerogenic DC are generally in an immature state exemplified bysuppressed expression of co-stimulatory molecules and IL-12. Variousagents have been used to inhibit maturation of DC in order to promotetolerance. These agents are used to generate DC that express lowerlevels of co-stimulatory molecules. The proteosome inhibitor PSI(N-benzyloxycarbonyl-Ile-Glu(O-tert-butyl)-Ala-leucinal) blocks NF-KBactivation and results in the in vitro production of tolerogenic DC(Yoshimura, S., et al., 2001. Eur J. Immunol. 2001 June;31(6):1883-93).N-acetylcysteine is an antioxidant which similarly blocks NF-KBactivation and generates immature, tolerogenic dendritic cells(Verhasselt. V., et al., 1999. J. Immunol. March 1;162(5): 2569-74).Vitamin D3 also inhibits dendritic cell maturation and leads toproduction of tolerogenic dendritic cells (Piemonti L., et al., 2000. J.Immunol. May 1;164(9):4443-51). A disadvantage of using such agents isthat there is no direct control of the resulting DC phenotype.Furthermore, DC exhibit plasticity in an in vivo environment which isdisadvantageous for using DC directly in immunotherapy. Therefore theability to generate DC with a specific phenotype and function would beadvantageous.

Post-Transcriptional gene silencing is a mechanism that functions toinhibit viral replication in many eukaryotic organisms (Hannon, G. J.2002. RNA Interference. Nature 418:244; Cogoni, C., et al., 2000. CurrOpin Genet Dev 10:638). This process is mediated by double stranded RNA(dsRNA) and can evoke many cellular reactions including the non-specificinhibition of protein synthesis seen in the interferon response ofmammalian cells (Levy, D. E. et al., 2001. Cytokine Growth Factor Rev12:143). It has recently been discovered that short sequences of RNAthat are 21 nucleotides in length (known as small interfering RNA orsiRNA) can bypass the broad suppression of the interferon response andcan lead to the specific degradation of cognate mRNA (Elbashir, S. M.,et al., 2001. Nature 411:494; Moss, E. G. 2001. Curr Biol 11 :R772).This process, known as RNA interference (RNAi), is specific as a singlesubstitution in the 21 nucleotide sequence can abrogate its effects, andis extremely efficient, since the siRNA is incorporated into anenzymatic complex that conducts multiple rounds of target mRNAdegradation (Tuschl, T. 2002. Nat Biotechnol 20:446). As such, RNAiprovides a useful tool for inhibiting endogenous gene expression, andcould provide a means to effectively modulate immune responses. Variousmethods of RNAi have been described for the altering gene expression inplant cells, drosophila and human melanoma cells as is described forexample in U.S. Patent Application No. 2002/0162126A1, PCT/US01/10188,PCT/EP01/13968 and U.S. Patent Application No. 2002/0173478A1.

In general, RNA interference has been found to be unpredictable with lowefficiency when used in vertebrate species (Fjose et al., Biotechnol.Annu. Rev. 7:31-57, 2001). Methods of RNA interference have not beenpreviously contemplated for use in the transformation of immune cellsand in particular the transformation of antigen presenting cells (APC)such as dendritic cells (DC) to produce a desired stable phenotype thatcan be further used in vitro, ex vivo and/or in vivo methods for themodulation of immune responses via the inhibition or stimulation of Tcell activity. Furthermore, immune cells specifically designed tosilence and thus suppress the expression of specific endogenous genes toaffect T cell functioning have not been previously contemplated, norcontemplated for use in methods of treating immune disorders.

SUMMARY OF THE INVENTION

The present invention provides immune cells that exhibit a targetedgene-specific knockout phenotype that can be used therapeutically tomodulate immune responses in a mammal. More specifically, the presentinvention provides altered DC that do not express one or more genesencoding a molecule involved in DC activity, and as such, suppress orstimulate immune system functioning via the modulation of T cellactivity.

The present invention also encompasses therapeutic methods for thetreatment of a variety of immune disorders with the use of the alteredDC. In embodiments of the invention, the DC may be transfected in vitroto produce a desired DC phenotype and then either used ex vivo oralternatively used in vivo as administered to a mammalian subject.

According to an aspect of the present invention there is provided amammalian immune cell that exhibits a targeted gene-specific knockoutphenotype, said immune cell being capable of altering an immune responsein a-mammal via the modulation of T cell activity. In embodiments, theimmune cell may be selected from an endothelial cell or an antigenpresenting cell (APC). In more preferred embodiments, the immune cellsis an APC selected from the group consisting of DC, macrophages, myeloidcells, B lymphocytes and mixtures thereof.

According to an aspect of the invention is a mammalian immune cellexhibiting a targeted endogenous gene-specific knockout phenotype, saidimmune cell altering an immune response in a mammal via the modulationof T cell activity

According to another aspect of the present invention is a mammalianimmune cell that exhibits a targeted gene-specific knockout phenotype,wherein said gene is selected from one or more of a surface marker, achemokine, a cytokine, an enzyme and a transcriptional factor.

According to another aspect of the present invention is an APC whichdoes not express one or more of a surface marker, a chemokine, acytokine, an enzyme and a transcriptional factor. In an embodiment ofthe invention, the APC is a DC.

According to another aspect of the present invention is a dendritic cell(DC) which contains at least one double-stranded RNA molecule capable ofinhibiting the expression of an endogenous target gene encoding amolecule selected from the group consisting of a surface marker, achemokine, a cytokine, an enzyme, a transcriptional factor andcombinations thereof.

According to another aspect of the present invention is a tolerogenicdendritic cell (DC) which contains at least one double-stranded RNAmolecule capable of inhibiting the expression of IL-12.

According to a further aspect of the invention is the use of a mammalianimmune cell that exhibits a targeted gene-specific knockout phenotype,wherein said gene is selected from one or more of a surface marker, achemokine, a cytokine, an enzyme and a transcriptional factor, in amedicament for the treatment of an immune disorder characterized byinappropriate T cell activity.

According to another aspect of the invention is the use of a siRNApossessing specific homology to part or the entire exon region of a geneencoding a surface marker, a chemokine, a cytokine, an enzyme or atranscriptional factor of an antigen presenting cell (APC), in amedicament for the treatment of an immune disorder characterized byinappropriate T cell activity.

According to yet another aspect of the invention is a composition forthe treatment of an immune disorder, said composition comprising atleast one of:

-   -   (a) a construct that inhibits the expression of an endogenous        target gene encoding a surface marker, a chemokine, a cytokine,        an enzyme or a transcriptional factor in an immune cell such        that said immune cell alters T cell activity; and    -   (b) an immune cell wherein said immune cell comprises at least        one construct that inhibits the expression of an endogenous        target gene encoding a surface marker, a chemokine, a cytokine,        an enzyme or a transcriptional factor, and    -   (c) a pharmaceutically acceptable carrier,    -   wherein said composition alters T cell activity leading to an        altered immune response.

According to another aspect of the invention is a method for inhibitingthe T cell activating ability of a DC, the method comprisingtransforming said DC with a constructcapable of inhibiting-theexpression of an endogenous target gene encoding a surface marker, achemokine, a cytokine, an enzyme or a transcriptional factor.

According to still a further aspect of the invention is a method fordecreasing the immunogenicity and rejection potential of an organ fortransplantation, said method comprising perfusing said organ with acomposition that suppresses T cell activity, said composition comprisingat least one construct that inhibits the expression of an endogenoustarget gene encoding a surface marker, a chemokine, a cytokine, anenzyme or a transcriptional factor and a pharmaceutically acceptablecarrier.

According to another aspect of the invention is a method for making animmune cell that alters the activity of T cells in vivo, said methodcomprising;

transforming immune cells in vitro with at least one construct thatinhibits the expression of an endogenous target gene encoding a surfacemarker, a chemokine, a cytokine, an enzyme or a transcriptional factor.

According to yet another aspect of the invention is method for thetreatment of autoimmune disorders and transplantation rejection in amammalian subject, said method comprising administering atherapeutically effective amount of a composition to said subject, saidcomposition comprising DC that contain at least one construct thatinhibits the expression of an endogenous target gene encoding a surfacemarker, a chemokine, a cytokine, an enzyme or a transcriptional factor,wherein said DC suppresses T cell activity.

According to another aspect of the invention is a method for thetreatment of autoimmune disorders and transplantation rejection in amammalian subject, said method comprising administering atherapeutically effective amount of a composition to said subject, saidcomposition comprising an siRNA targeted to inhibit expression of anendogenous target gene in an antigen presenting cell, said gene encodinga surface marker, a chemokine, a cytokine, an enzyme or atranscriptional factor, wherein said siRNA suppresses T cell activity.

In aspects of the invention the construct may be any suitable constructthat can be used to target and silence a particular gene of interest. Inembodiments, the construct is siRNA or hybrid DNA/RNA provided alone orwithin a suitable vector or plasmid.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating embodiments of the invention are given by wayof illustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be describedmore fully with reference to the accompanying drawings:

FIG. 1 shows the efficacy of DC siRNA transfection. Day 7 bone marrowderived DC (1×10⁶) were transfected with unlabeled control siRNA(Ctrl-siRNA, left), or fluorescein labelled siRNA specific forluciferase GL2 duplex (Fl-siRNA, middle) at 60 pM concentration.Fl-siRNA was also added to day 4-cultured DC without transfectionreagents (Phagocytosis, right). DC were activated with LPS/TNFα on day 8and the transfection efficacy was assessed by flow cytometry on day 9.Data are representative of three independent experiments.

FIG. 2 shows that DC viability is not affected by siRNA transfection. DCcultured from bone marrow progenitors and 1×10⁶ day-7 immature DC wereleft untreated or were transfected with GenePorter alone, siRNA-IL12p35alone, or the combination of both for 48 hrs. Percentage apoptosis andnecrosis was assessed using annexin-V and propidium iodine (PI),respectively, by flow cytometry. Data are representative of threeindependent experiments.

FIG. 3 shows that siRNA transfection of DC does not alter nor induce DCmaturation. In panel 3A immature DC (1×10⁶) were cultured alone(untransfected), pre-treated for 24 hrs with GenePorter (mocktransfected), or transfected with 60 pM siRNA-IL12p35. The transfectedDC were subsequently activated for 24 hrs with 10 ng/ml LPS and 10 ng/mlTNF-α. Maturation was assessed by expression of CD11c, MHC II, CD40, andCD86 by flow cytometry using FlTC-conjugated antibodies (solid line),and isotype controls (broken line). In panel 3B immature DC (1×10⁶) wereuntreated (untransfected), treated with GenePorter alone (mocktransfected) or transfected with 60 pM siRNA-IL12p35 for 24 hrs at whichtime maturation was assessed by expression of CD11c, MHC II, CD40, andCD86 by flow cytometry using FITC-conjugated antibodies (solid line),and isotype controls (broken line). Data are representative of threeindependently performed experiments.

FIG. 4 shows the specificity of gene inhibition by siRNA. DC (1×10⁶)were transfected with 60 pM siRNA-IL12p35, siRNA-IL12p40 or Geneporteralone (mock transfected). The transfected DC were activated with 10ng/ml LPS and 10 ng/ml TNF-α for 24 hrs. RNA from the treated DC wasextracted by the Trizol method. RT-PCR was performed to assessexpression of IL-12p35, IL-12p40 and GAPDH using primers described inthe examples section. Data are representative of three independentexperiments.

FIG. 5 shows that siRNA-IL12p35 transfection of DC specifically blocksIL-12 and upregulates IL-10. DC (1×10⁶) were unmanipulated (control),transfected with Geneporter alone (mock transfected), transfected with60 pM siRNA-IL12p35, or 60 pM siRNA-IFNγ (siRNA control). Thetransfected DC were activated with 10 ng/ml LPS and 10 ng/ml TNFα for 24hrs. In panel 5A the supernatants were harvested from cultures andanalyzed for IL12 p70 production using ELISA. In panel 5B thesupernatants were harvested from cultures and analyzed for IL-10production using ELISA. Data represent mean+SEM and are representativeof three experiments (*, p<0.01; by one-way ANOVA and Newman-Keulstest).

FIG. 6 shows that siRNA-IL12p35 transfection inhibits DC allostimulatoryability. C57BL/6 derived DC (1×10⁶) were untreated (untransfected, 0),transfected with GenePorter alone (mock transfected, 0), transfectedwith 60 pM siRNA-IFNγ (control siRNA, Δ) or transfected with 60 pMsiRNA-IL12p35 (•) for 24 hrs. Allogeneic (BALB/c) T cells (2×10⁵/well)were incubated with siRNA-treated DC at the indicated numbers for 72hrs. Proliferation was determined using [³H]-thymidine incorporation.Data are representative of three independent experiments. (*p<0.01; byone-way ANOVA and Newman-Keuls test).

FIG. 7 shows that siRNA-IL12p35-transfected DC promote Th2 polarization.In panel 7A C57/BL6 bone marrow derived DC were pretreated withGenePorter alone (mock transfected), transfected with 60 pMsiRNA-lL12p35 for 24 hrs. Subsequently siRNA-treated DC (10⁶) werecultured with allogeneic (BALB/c) T cells (10×10⁶) for 48 hrs. T cellswere purified from co-culture using a T cell column and RT-PCR wasperformed for IL-4, IFN-γ, and GAPDH. In panel 7B C57/BL6 bone marrowderived DC were unmanipulated (control), pretreated with GenePorteralone (mock transfected), transfected with 60 pM siRNA-IL12p35, or 60 pMsiRNA-IFN-γ (siRNA control) for 24 hrs. siRNA-treated DC (10⁶) weresubsequently cultured with allogeneic (BALB/c) T cells 10×10⁶) for 48hrs. Supematants were collected from the cultures and IFN-γ (Th1cytokine) and IL-4 (Th2 cytokine) production was assessed by ELISA.(*p<0.01; by one-way ANOVA and Newman-Keuls test).

FIG. 8 shows that siRNA-IL12p35-treated DC stimulate antigen-specificTh2and inhibit Th1 responses in vivo. Day 7 bone marrow derived DCcultured in GM-CSF and IL-4 were transfected with IL12p35-siRNA, or mocktransfected. Subsequently cells were pulsed with 10 μg/ml of KLH for 24hrs and injected subcutaneously (5×10⁵ cells/mouse) into syngeneicC57BL/6 mice. After 10 days, T cells from lymph nodes were isolated fromrecipient mice. A KLH-specific recall response was performed asdescribed in the example section. IFN-γ and IL-4 response to KLH wasassessed by ELISA. Data shown are pooled from 3 independent experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides transformed immune cells that exhibit agene specific targeted knock-out phenotype. Such transformed immunecells can be used in a variety of therapeutic in vitro, ex vivo and invivo methods to modulate T cell activity and thus have use intherapeutic approaches for the treatment of immune disorders inmammalian subjects.

The immune cells of the invention exhibit a targeted gene-specificknockout phenotype which imay be accomplished using any technique thatprovides for the targeted silencing of an endogenous gene. In one aspectof the invention the technique of RNAi (RNA interference) was used tocreate transformed immune cells suitable for use for the modulation of Tcell activity in vitro, ex vivo or in vivo. In this aspect, the immunecells are transfected with a siRNA (small interfering RNA) designed totarget and thus to degrade a desired mRNA in order not to express theencoded protein that is involved in T cell activity. Thus suchtransfected immune cells may be used to suppress or stimulate immunesystem functioning via the modulation of T cell activity. It isunderstood by those of skill in the art that any method for silencing aspecific gene may be used in the present invention. Representativeexamples of suitable techniques include but are not limited to RNAi andhybrid DNA/RNA constructs. The hybrid DNA/RNA constructs are essentiallysiRNA constructs in which the nucleic acid composition used forsilencing is altered to include DNA (Lamberton J. and Christian A. 2003.Mol. Biotechnol. June;24(2):111-20, the entirety of the disclosure isincorporated herein by reference).

It is desirable to modulate T cell activity, ie. suppress T cellactivity in a variety of immune disorders selected but not limited tothe group consisting of septic shock, rheumatoid arthritis, transplantrejection, scleroderma, immune mediated diabetes, chronic inflammatorybowel syndrome, HIV, cancer, colitis, Crohn's disease, Goodpasture'ssyndrome, Multiple Sclerosis, Grave's disease, Hashimoto's thyroditis,Autoimmune pernicious anemia, Autoimmune Addison's disease, Vitiligo,Myasthenia gravis, Scleroderma, Systemic lupus erythematosus, PrimarySjogren's syndrome, Polymyositis, Pemphigus vulgaris, Ankylosingspondylitis, Acute anterior uveitis, Hypoglycemia and inflammationassociated with chronic illness. Thus the siRNA, transfected immunecells and compositions containing such can be used in methods to treatthe aforementioned immune disorders by the down regulation of T cellactivity leading to a prevention or decrease in an autoimmune responseand prevention of tissue/organ rejection.

Immune cells for use in the present invention may be selected fromantigen presenting cells (APC) and endothelial cells. Both APC andendothelial cells (Limmer A., et al., 2001. Arch Immunol Ther Exp(Warsz). Suppl 1:S7-11; Perez V/L., et al., 1998. Cell Immunol. October10;189(1):31-40) are known to be able to activate T cells. In preferredembodiments of the invention, the immune cells are APC that may beselected from the group consisting of macrophages, myeloid cells, Blymphocytes, DC and mixtures thereof. It is also within the scope of thepresent invention to use other APC capable of activating T cells throughthe T cell receptor as is understood by one of skill in the art. Inparticularly preferred embodiments of the invention, the immune cell isa DC. APC such as DC are known to be phagocytic in nature and thus tendto take up molecules within their environment. In the present inventionDC is specifically demonstrated to be successfully altered with siRNA toexhibit a stable phenotype. Therefore one of skill in the art wouldreadily understand that any APC may be altered in accordance with thepresent invention and used in the methods of the invention. It is alsounderstood that a combination of different types of immune cells may beused in the methods of the present invention.

According to an embodiment of the invention, DC are transformed with adesigned siRNA. In this embodiment DC must be isolated from a subjectand expanded in vitro. DC are typically derived from a source such asbone marrow, peripheral blood, spleen and lymph. Blood is the preferredsource of DC because it is readily accessible and may be obtained inlarge quantities. Substances which stimulate hematopoiesis (i.e. G-CSFand GM-CSF) may be first administered to the subject in order toincrease the number of DC. Blood is treated to isolate the DC from othercell types by standard methods known in the art. Isolated DC cultured invitro may be treated with cytokines to increase their number. Methodsfor isolating and ex vivo culture of DC are known in the art anddescribed for example in U.S. Pat. Nos. 5,199,942, 5,851,756, 6,017,527,6,251,665, 6,458,585 and 6,475,483 (the disclosures of which areincorporated herein by reference in their entirety).

The present invention also encompasses therapeutic methods for thetreatment of a variety of immune disorders in a mammalian subject. Themethods may involve the use of a siRNA designed for use directly in vivoto block the expression of a gene by an immune cell, the gene expressinga protein involved in the activity of T cells which elicits an immunedisorder. Alternatively, the methods may involve the use of an immunecell which contains at least one double-stranded RNA molecule (siRNA)that inhibits the expression of an endogenous target gene encoding asurface marker, a chemokine, a cytokine, an enzyme or a transcriptionalfactor. In preferred embodiments of the invention, the methods of theinvention comprise the use of an altered (i.e. transformed) DC thatcontains a double-stranded RNA molecule that inhibits the expression ofan endogenous target gene encoding a surface marker, a chemokine, acytokine, an enzyme or a transcriptional factor. Still in otherembodiments, the therapeutic method may involve ex vivo treatment oftissues and/or organs intended for transplantation. In aspects of theinvention, the siRNA possesses specific homology to part or to theentire exon region of a surface marker, a chemokine, a cytokine, anenzyme or a transcriptional factor normally expressed by the immune cellsuch that the gene is silenced

It is understood by one of skill in the art that the siRNA as hereindescribed may also include altered siRNA that is a hybrid DNA/RNAconstruct or any equivalent thereof.

In preferred embodiments of the invention the transfected DC cells areprepared by the method of RNAi. RNA interference is a mechanism ofpost-transcriptional gene silencing. Specific gene silencing is mediatedby short strands of duplex RNA of approximately 21 nucleotides in length(termed small interfering RNA or siRNA) that target the cognate mRNAsequence for degradation. While many techniques have been used to blockspecific molecules in vitro and in vivo, such as anti-senseoligonucleotides (Gerwitz, A. M. 1999. Curr Opin Mol Ther 1:297) andmonoclonal antibodies (Drewe, E., et al., 2002. J Clin Pathol 55:81),RNAi was used in the present invention because it provides severaldistinct advantages. First, mRNA degradation by siRNA is extremelyefficient as only a few copies of dsRNA are necessary to activate theRNA Induced silencing complex (RISC) (Martinez, J. A. et al., 2002. Cell10:563). Once RISC is activated it can conduct multiple rounds ofgene-specific mRNA cleavage. Second, RNAi is specific, in that onlysequences with identity to one of the strands of dsRNA will be cleaved(Hannon, G. J. 2002. Nature 418:244). Third, the RNAi effect is longlasting and can be spread to progeny cells after replication, although adilution effect is evident in mammalian cells (Fire, A., et al., 1998.Nature 391:806). This technique is relatively simple, giving rise to anin vitro knock down phenotype within days that can be confirmed withmany antibody based detection systems (such as ELISA or WesternBlotting), or if an antibody is not available, by RT-PCR or functionalassays.

DC may be transformed with siRNA alone, siRNA contained within a plasmidor vector that results in the production of the siRNA, siRNA containedwithin a plasmid or vector that further expresses a selected antigen andsiRNA together with a mRNA from a tumor cell. In the case of the plasmidor is vector further expressing a selected antigen, the DC will processor modify the antigen in a manner-to promote the stimulation of T cellactivity by the processed or modified antigens. Methods for making siRNAand cell transformation are described for example in U.S. PatentApplication 2002/0173478, U.S. Patent Application 2002/0162126,PCT/US01/10188, PCT/EP01/13968 and in Simeoni F., et al., 2003 NucleicAcids Res June 1;31(11):2717-24 (the disclosures of which areincorporated herein in their entirety). Methods for producing antigenpulsed DC are known and exemplified for example in U.S. Pat. No.6,497,876 and U.S. Pat. No. 6,479,286 (the disclosures of which areincorporated herein by reference in their entirety). Methods for makingsiRNA plasmids or vectors are also known and described for example inU.S. Patent Application 2003/0104401, in Morris M. C., et al., 1997.Nucleic Acid Res. July 15:25(14):2730-6 and in Van De Wetering M., etal., 2003, EMBO June;4(6):609-15 (the disclosures of which areincorporated herein in their entirety). Suitable lipid-based vectors mayinclude but are not limited to lipofectamine, lipofectin, oligofectamineand GenePorter™. Methods for producing tumor derived RNA for pulsing DCare also known to those of skill in the art and are described forexample in U.S. Patent Application 2002/0018769 (the disclosure of whichis incorporated herein in its entirety).

In embodiments of the invention, DC are transformed to contain adouble-stranded RNA molecule that inhibits the expression of anendogenous target gene encoding a protein that either suppresses T cellactivation or alternatively stimulates T cell activation. For thesuppression of T cell activation, the immune cells of the invention aretransformed with a double-stranded RNA molecule that inhibits theexpression of a gene that encodes a co-stimulatory molecule, cytokine,adhesion molecule, enzyme or transcription factor. Representativeexamples of such co-stimulatory molecules, cytokines, adhesionmolecules, enzymes and transcription factors may be selected from thegroup consisting of TNFα, IL-1, IL-1b, IL-2, TNFβ, IL-6, IL-7, IL-8,IL-23, IL-15, lL18, IL-12, IFNγ, IFNα, lymphotoxin, DEC-25, CD11c, CD40,CD80, CD86, MHCI, MHCII, ICAM-1, TRANCE, CD200, CD200 receptor, CD83,CD2, CD44, CD91, TLR-4, TLR-9, 4-1 BBL, nicotinic receptor, GITR-L,OX-40L, CD-CK1, TARC/CCL17, CCL3, CCL4, CXCL9, CXCL10, IKK-β, NF-κB,STAT4, ICSBP/IFN, regulatory factor 8, TRAIL, Inos, arginase, FcgammaRIand II, thrombin, MIP-1α and MIP-1B.

For the activation of T cells where such activation is desired, theimmune cells of the invention are transformed with a double-stranded RNAmolecule that inhibits the expression of a gene encoding a surfacemarker or enzyme that suppresses T cell activation. Representativeexamples of such surface markers and enzymes may be selected from thegroup consisting of B7-H1, EP2, IL-10 receptor, VEGF-receptor, CD101,PD-L1, PD-L2, HLA-11, DEC-205, CD36 and indoleamine 2,3-dioxygenase. Itmay be desirable to activate T cells in a variety of conditionsassociated with immune suppression such as but not limited to cancer,HIV and parasitic infections. Where immune suppression is present, it isdesirable to use the cells and methods of the invention to increase Tcell activity leading to an enhanced immune response (Curiel T. J., et.Al., 2003. Nat Med May;9(5):562-7).

It is within the scope of the invention to transform a selected immunecell with more than one double-stranded RNA molecule (an siRNA) orhybrid DNA/RNA in order to simultaneously inhibit the expression of morethan one endogenous gene normally expressed by the immune cell. Thenumber of double-stranded RNA molecules transformed into any givenimmune cell being dependent on the resultant extent of inhibition of theexpression of the target gene which is readily determined as isunderstood by one of skill in the art.

In the present invention in one embodiment, the induction of RNAi in DCwas conducted using siRNA specific for IL-12 p35 (siRNA-IL12p35). It wasdemonstrated that bioactive IL-12 p70 production in bone marrow-derivedDC was inhibited after stimulation with LPS and TNF-α, and wasaccompanied by an increase in IL-10 production. Moreover, whensiRNA-IL12p35-treated DC were cultured with allogeneic T cells, a Th2polarization was observed since T cell expression of IFN-γ was reducedwhile IL-4 was increased. Inhibiting IL-12 production usingsiRNA-IL12p35 was associated with suppressed DC allostimulatoryfunction. In vivo, initiation of antigen-specific Th2 responses wasobserved when DC treated with siRNA-IL12p35 were pulsed with KLH andused for immunization experiments. Overall these results demonstrate forthe first time that RNAi can be induced in DC and that siRNA is a potenttool for modulating DC function and subsequently T cell polarization.

DC are Efficiently Transfected with siRNA

To establish a protocol for RNAi in DC, the siRNA-transfection efficacywas first assessed. Many studies have shown a limited ability of DC tobe transfected with DNA. To determine the transfection efficacy,fluorescein labelled siRNA was synthesized that is specific forluciferase (FL-siRNA-Luc), a gene that does not exist in mammalian cellsand thus does not affect cellular function. siRNA lacking fluorescein(siRNA-Luc) was used as a non-labelled control. FL-siRNA-Luc andsiRNA-Luc were transfected by GenePorter into bone marrow-derived andcultured DC. After 24 hrs siRNA transfection, the percentages of DC thathad incorporated FL-siRNA-Luc were quantified by flow cytometry. As seenin FIG. 1, FL-siRNA-Luc had been successfully incorporated into 88% ofthe cells, as analyzed by flow cytometry.

It was then assessed whether immature DC are able to internalize nakedsiRNA. Immature DC on day 4 were cultured with FL-siRNA-Luc in theabsence of transfection reagent, and assessed for siRNA internalizationby flow cytometry on day 9 of culture. Despite the long incubationperiod, 19% of DC still contained incorporated siRNA (FIG. 1),suggesting that naked siRNA may be used for transfection of DC.

siRNA Transfection does not Alter DC Viability, Maturation or Phenotvpe

One of the major concerns for gene transfection is that transfectionreagents may affect cellular function or viability. Although a highlevel of transfection efficiency was already demonstrated using theGenePorter method, it was further needed to establish whether siRNA orthe transfection procedure itself altered the viability of the DC. Thus,day-7 bone marrow-derived DC were treated with transfection reagent(GenePorter) alone, siRNA-lL12p35 alone, or the combination oftransfection reagent and siRNA-IL12p35. After 24 hrs of transfection,apoptosis and necrosis was assessed using annexin-V and propidium iodine(PI) staining respectively. Compared to untreated DC, neither thetransfection protocol alone, nor the siRNA affected cell viability (FIG.2).

Next it was addressed whether the siRNA or the transfection procedureaffected DC maturation. DC were transfected with siRNA followingactivation with LPS and TNF-α. DC maturation was assessed by flowcytometry to analyze expression of MHC II, CD40, and CD86 or theDC-specific marker CD11c. It can be seen that neither treatment withsiRNA nor mock transfection altered DC maturation in response to LPS andTNF-α (FIG. 3A).

An additional concern associated with transfecting DC with nucleic acidsis induction of maturation. Since long double stranded RNA(poly(I):poly(C)) has previously been shown to induce maturation andactivation of immature DC (25), it was determined whether or not siRNAhad the same effect. Thus, immature DC were treated with siRNA-IL12p35for 24 hrs and cell surface maturation markers were assessed by FACS.FIG. 3B illustrates that siRNA treatment alone failed to upregulate MHCII, CD40, or CD86 on immature DC. Although these experiments used aconcentration of 60 pM of siRNA-IL12p35, higher concentrations ofsiRNA-IL12p35 (up to 10 fold) were also assessed, with no alteration inviability or differentiation (data not shown). These data indicate thattransfection of DC with siRNA-IL12p35 affects neither the viability norphenotype.

siRNA induces Specific Gene Silencing in DC

The specificity of siRNA induced gene silencing in DC was examined bytransfecting DC with siRNA-IL12p35 and siRNA targeted to the p40component of IL-12 (siRNA-IL12p40). Transcripts of IL-12 p35 and IL-12p40 were detected by RT-PCR using primers flanking the siRNA targetedsequence. Specific inhibition was demonstrated at the transcript level:siRNA-IL12p35 exclusively suppressed p35 transcripts while siRNA-IL12p40suppressed only p40 transcripts (FIG. 4). In addition, bothsiRNA-IL12p35 and siRNA-IL12p40 failed to affect transcripts of thehouse-keeping gene GAPDH. These data suggested that siRNA-mediated genesilencing is specific in DC.

siRNA-IL12p35 Inhibits IL-12 Expression in DC

It was verified whether siRNA-IL12p35 can block production of IL-12protein. Since IL-12p35 is critical for the formation of the IL-12 p70heterodimer, the production of this cytokine was assessed in thesupernatant of LPS/TNF-α-activated DC using ELISA. DC transfected withsiRNA-IL12p35 were stimulated with LPS and TNF-α for 48 hrs to inducematuration and cytokine expression. To confirm specificity of genesilencing, siRNA specific for IFN-γ (siRNA-control) was used since thiscytokine is not expressed in bone marrow derived DC. Additionally,negative controls included DC transfected with GenePorter alone (mocktransfected DC) and unmanipulated DC (untreated control). As shown inFIG. 5A, siRNA-IL12p35 reduced IL-12p70 heterodimer production (asdetermined by ELISA) by 85-90% compared to untreated or mock transfectedDC. More importantly this effect was specific since no significantdifference in IL-12p70 production was seen in DC treated with the IFN-γsiRNA-control. In addition, levels of IL-10 production were tested sincea reciprocal relationship with IL-12 production has been previouslyreported (27). IL-10 production in DC treated with siRNA-IL12p35 wassignificantly and specifically upregulated compared to controls (FIG.5B).

siRNA-IL12D35 Suppresses DC Allostimulatory Activity

DC function can be characterized in part by their ability to stimulatealloreactive T cells in the mixed lymphocyte reaction (MLR) (8). Todetermine whether siRNA-IL12p35 affected DC allostimulatory activity,MLR was performed using DC transfected with siRNA-IL12p35,siRNA-control, mock transfected, or untreated controls. Allogeneic Tcells were cultured with siRNA-transfected DC for 48 hrs at which pointallostimulation was determined by proliferation. While the control DCgroups all showed similar allostimulatory activity, DC transfected withsiRNA-IL12p35 significantly suppressed this response (FIG. 6).

siRNA-lL12p35 Treated DC Promote Th2 Differentiation

Since IL-12p70 is a key cytokine responsible for polarizing T cellstowards an IFN-γ-producing or Th1 phenotype (Trinchieri, G. 1998. AdvImmunol 70:83), it was assessed whether allostimulation with DC thatwere transfected with siRNA-IL12p35 could alter cytokine production fromresponding T cells. Mock transfected DC stimulated high IFN-γ and lowIL4 mRNA transcripts from responding T cells, however, stimulation withsiRNA-IL12p35 treated DC resulted in low IFN-γ and high IL-4 transcripts(FIG. 7A). To confirm these results at the protein level IFN-γ and IL-4were assayed from MLR culture supernatants using ELISA. The T cellsincubated with siRNA-IL12p35-treated DC produced low levels of IFN-γ(FIG. 7B) and high levels of IL-4 (FIG. 7C). In contrast, T cellsincubated with untransfected DC, GenePorter transfected DC or DCtransfected with control siRNA showed a cytokine profile of high IFN-αand low IL-4. These data suggest that siRNA-IL12p35-treated DC have theability to polarize naïve T cells along the Th2 pathway.

Modulation of Antigen-Specific Response In Vivo using siRNA-IL12p35Treated DC

Although a shift from Th1 cytokine production to Th2 is seen when naïveT cells are incubated with siRNA-IL12p35-treated DC, it was investigatedwhether this effect could also be obtained in vivo. To accomplish this,siRNA-IL12p35-treated or mock transfected DC with KLH were transfectedand used as immunogens in vivo by injecting into syngeneic hosts.Ten-days after immunization with KLH-pulsed control DC, a Th1 recallresponse was evident when draining lymph node cells from recipient micewere challenged with KLH in vitro, as determined by upregulated IFN-γand downregulated IL-4 production (FIG. 8). Under the same conditionsthe siRNA-IL12p35-treated DC promoted a Th2 shift in the recall cytokineresponse, showing increased IL-4 production and suppressed IFN-γ. Theseresults suggest that antigen-pulsed and siRNA-modified DC can be used tomodulate the Th1 vs Th2 balance in vivo during a primary immuneresponse.

Interestingly, DC silenced by siRNA-IL12p35 showed decreasedallostimulatory capacity which is in contrast to results reported usingDC generated from IL-12 knockout mice that possess normalallostimulatory activity (Piccotti, J. R., et al., 1998. J Immunol160:1132; Tourkova I. L., et al., 2001. Immunol Lett 78:75). Weattribute this discrepancy to compensatory immunological mechanisms thatmay have arisen in the lifetime of the IL-12 knockout mice. This issuggested by studies that have demonstrated the importance of IL-12 inMLR. First, IL-12 production by antigen presenting cells wasdemonstrated to be critical for MLR proliferative response sinceaddition of anti-IL-12 antibodies resulted in suppression ofproliferation (Kohka, H., et al., 1999. J Interferon Cytokine Res19:1053). Second, overexpression of IL-12 in DC results in increasedallostimulatory function (Kelleher, P., et al., 1998. Int Immunol10:749). Another possible explanation for suppressed MLR insiRNA-IL12p35-transfected DC is that the increased IL-10 production mayact as an inhibitor of T cell proliferation (Wang X. N., et al., 2002.Transplantation 74:772; Tadmori W., et al., 1994. Cytokine 6:462). Otherstudies examining naturally occurring Th2-promoting DC have shown thatthese cells have a reduced allostimulatory function and reduced IL-12production (Gao J. X., et al., 1999. Immunology 98:159; Khanna A., etal., 2000. J Immunol 164:1346). The combination of Th2 promotingproperties, as well as poor allostimulation suggests that siRNA-IL12p35transfected DC may possess the phenotype of a “tolerogenic” DC and thusmay be useful for treatment of Th1 mediated autoimmune diseases andtransplant rejection.

The present invention provides methods of using therapeutic compositionscomprising siRNA designed to target a specific mRNA as well as activatedand nonactivated altered (i.e.transformed) immune cells that contain thesiRNA in embodiments as described supra. A feature of DC is theircapacity to migrate or home to T-dependent regions of lymphoid tissueswhere DC may affect T cell activity and elicit a modulated immuneresponse. Therefore, in vivo administration of a siRNA composition wouldbe effective in targeting and having a modulatingeffect on T cellactivity.

In one embodiment, the compositions comprise DC containing siRNAspecifically designed to degrade mRNA encoding a surface marker, achemokine, a cytokine, an enzyme or a transcriptional factor such thatthe transformed DC leads to a lack of expression of the surface marker,chemokine, cytokine, enzyme or transcriptional factor and as a resultaffect the activity of T cells to modulate an immune response. Such DCmay be provided as compositions for administration to a mammaliansubject or as compositions for ex vivo approaches for the treatment ofcells, tissues and/or organs for transplantation. Such compositions maycontain pharmaceutically acceptable carriers or excipients suitable forrendering the mixture administrable orally or parenteraly,intravenously, intradermally, intramuscularly or subcutaneously ortransdermally. The transformed immune cells or siRNA may be admixed orcompounded with any conventional, pharmaceutically acceptable carrier orexcipient as is known to those of skill in the art.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the compositions of this invention,its use in the therapeutic formulation is contemplated. Supplementaryactive ingredients can also be incorporated into the pharmaceuticalformulations.

It will be understood by those skilled in the art that any mode ofadministration, vehicle or carrier conventionally employed and which isinert with respect to the active agent may be utilized for preparing andadministering the pharmaceutical compositions of the present invention.Illustrative of such methods, vehicles and carriers are those described,for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970), thedisclosure of which is incorporated herein by reference. Those skilledin the art, having been exposed to the principles of the invention, willexperience no difficulty in determining suitable and appropriatevehicles, excipients and carriers or in compounding the activeingredients therewith to form the pharmaceutical compositions of theinvention.

It is also understood by one of skill in the art that the compositionsof the invention may be provided on a device for in vitro, ex vivo or invivo use. Suitable structures may include but are not limited to stents,heart valves, implants and catheters.

The therapeutically effective amount of active agent to be included inthe pharmaceutical composition of the invention depends, in each case,upon several factors, e.g., the type, size and condition of the patientto be treated, the intended mode of administration, the capacity of thepatient to incorporate the intended dosage form, etc. Generally, anamount of active agent is included in each dosage form to provide fromabout 0.1 to about 250 mg/kg, and preferably from about 0.1 to about 100mg/kg.

While it is possible for the agents to be administered as the rawsubstances, it is preferable; in view of their potency, to present themas a pharmaceutical formulation. The formulations of the presentinvention for mammalian subject use comprise the agent, together withone or more acceptable carriers therefor and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. Desirably, theformulations should not include oxidizing agents and other substanceswith which the agents are known to be incompatible. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing into association the agent with the carrier, whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the agent with the carrier(s) and then, if necessary,dividing the product into unit dosages thereof.

Formulations suitable for parenteral administration convenientlycomprise sterile aqueous preparations of the agents, which arepreferably isotonic with the blood of the recipient. Suitable suchcarrier solutions include phosphate buffered saline, saline, water,lactated ringers or dextrose (5% in water). Such formulations may beconveniently prepared by admixing the agent with water to produce asolution or suspension, which is filled into a sterile container andsealed against bacterial contamination. Preferably, sterile materialsare used under aseptic manufacturing conditions to avoid the need forterminal sterilization.

Such formulations may optionally contain one or more additionalingredients among which may be mentioned preservatives, such as methylhydroxybenzoate, chlorocresol, metacresol, phenol and benzalkoniumchloride. Such materials are of special value when the formulations arepresented in multidose containers.

Compositions of the invention comprising a selected targeting siRNA canalso comprise one or more suitable adjuvants. In this embodiment siRNAcan be used as a vaccine in order to stimulate or inhibit T cellactivity and polarize cytokine production by these T cells. As is wellknown to those of ordinary skill in the art, the ability of an immunogento induce/elicit an immune response can be improved if, regardless ofadministration formulation (i.e. recombinant virus, nucleic acid,peptide), the immunogen is coadministered with an adjuvant. Adjuvantsare described and discussed in “Vaccine Design—the Subunit and AdjuvantApproach” (edited by Powell and Newman, Plenum Press, New York, U.S.A.,pp. 61-79 and 141-228 (1995). Adjuvants typically enhance theimmunogenicity of an immunogen but are not necessarily immunogenic inand of themselves. Adjuvants may act by retaining the immunogen locallynear the site of administration to produce a depot effect facilitating aslow, sustained release of immunizing agent to cells of the immunesystem. Adjuvants can also attract cells of the, immune system to animmunogen depot and stimulate such cells to elicit immune responses. Assuch, embodiments of this invention encompass compositions furthercomprising adjuvants.

Desirable characteristics of ideal adjuvants include:

-   -   1) lack of toxicity:    -   2) ability to stimulate a long-lasting immune response;    -   3) simplicity of manufacture and stability in long-term storage;    -   4) ability to elicit both cellular and humoral responses to        antigens administered by various routes, if required:    -   5) synergy with other adjuvants;    -   6) capability of selectively interacting with populations of        antigen presenting cells (APC);    -   7) ability to specifically elicit appropriate Tr, TR1 or TH2        cell-specific immune responses; and    -   8) ability to selectively increase appropriate antibody isotype        levels (for example, IgA) against antigens/immunogens.

Suitable adjuvants include, amongst others, aluminium hydroxide,aluminium phosphate, amphigen, tocophenols, monophosphenyl lipid A,muramyl dlpeptide and saponins such as Quill A. Preferably, theadjuvants to be used in the tolerance therapy according to the inventionare mucosal adjuvants such as the cholera toxine B-subunit or carbomers,which bind to the mucosal epithelium. The amount of adjuvant dependingon the nature of the adjuvant itself as is understood by one of skill inthe art.

Compositions of siRNA of the present invention may also be providedwithin antibody labelled liposomes (immunoliposomes) or antibody-doublestranded RNA complexes. In this aspect, the siRNA is specificallytargeted to a particular cell or tissue type to elicit a localizedeffect on T cell activity. Specifically, the liposomes are modified tohave antibodies on their surface that target a specific cell or tissuetype. Methods for making of such immunoliposomal compositions are knownin the art and are described for example in Selvam M. P., et.al., 1996.Antiviral Res. December;33(1):11-20 (the disclosure of which isincorporated herein in its entirety).

In one representative embodiment of the invention, siRNA to TNFα is madeaccording to the methods of Tuschl T., et al., 1999. Genes Dev.13:3191-97 and Tuschl T., et.al., 1998. EMBO J. 17:2637-2650. In thesemethods, 21 nucleotide base-pair sequences are chemically synthesizedusing a new 5′-silyl protecting group in conjunction with a uniqueacid-labile 2′-orthoester protecting group, 2′-bis(acetoxyethoxy)-methylether (2′-ACE). The 2′-protecting groups are rapidly and completelyremoved under mild conditions in aqueous buffers. This “2′-ACE™technology (Dharmacon Inc. CO, USA) enables the synthesis of RNAoligonucleotides in high yield. To the siRNA specific to TNFα is admixedan agent that crosses the cell membrane and enters the nucleus in orderto achieve maximal inhibition of TNFα. Such agents are known to those ofskill in the art and may be selected from cationic and anionic liposomesas well as compositions of chemicals which permit transmembrane entranceof the siRNA without affecting the function of the nucleotides. Inaddition to compounds which allow entry of siRNA into the cell, thesiRNA may be mixed with pharmaceutically acceptable carriers asdescribed supra.

The composition containing the siRNA may be administered to a mammaliansubject by a variety of methods described supra. The optimal route ofadministration is dependent upon the area of the body where suppressionof TNFα is most desired. For diseases associated with systemic rises inTNFα, the dosage of siRNA administered can be guided by serum ELISAmeasurements for levels of this cytokine. In mammalian subjects wheresystemic intravenous administration is desired, siRNA can be infused viaa portable volumetric infusion pump at a rate between about 1-6 mL/hourdepending on the volume to be infused as is understood by one of skillin the art. Doses of 0.1 mg/kg/day to about 10 mg/kg/day may beadministered for a time period necessary to suppress TNFα expression.

Suppression of the cytokine TNFα is desirable in a variety of immunedisorders that include but are not limited to septic shock, rheumatoidarthritis, transplant rejection, scleroderma, immune mediated diabetes,chronic inflammatory bowel syndrome, HIV, cancer, colitis, Crohn'sdiseaseand inflammation associated with chronic illness. It is desirableto suppress the expression of a molecule on an immune cell such as acytokine involved in a particular immune related disorder. As such, theinvention is applicable to the treatment of a variety of immunedisorders associated with the expression of surface markers, enzymes,cytokines, chemokines and transcription factors on an immune cell suchas a DC leading to a desired decrease in T cell activity and thusalleviating the immune condition. For the treatment of autoimmunedisorders using transformed immune cells of the invention, it isdesirable to use the mammalian subjects own cells for transformation andreintroduction into the subject for therapy.

In another embodiment of the invention, the siRNA and/or altered immunecells, in particular DC that exhibits a targeted gene-specific knockoutphenotype, can be used in compositions to perfuse cells, tissues and/ororgans ex vivo for transplantation. In this aspect, mammalian donortissues and/or organs are perfused ex vivo with a siRNA composition ortransformed immune cell composition of the invention prior totransplantation into a mammalian host. In this manner, the tissue ororgan is less susceptible to rejection in the host as T cell activity issuppressed. Methods of tissue/organ perfusion using perfusion machinesfor example are known to those of skill in the art.

In another embodiment, the invention provides methods for generatingtolerogenic dendritic cells (DC) as for example by the suppression ofexpression of IL-12 on DC using RNAi. Such tolerogenic DC can be used inmethods for the treatment of autoimmune disorders where the antigen isknown. DC can be isolated from a mammalian subject from bone marrow orperipheral blood and loaded with the autoantigen. These DC are thenadministered siRNA directed to IL-12 suppression as described supra orin the examples section and then re-infused into the mammalian subject.These DC only generate T regulatory cells and/or Th2 cells specific forthe autoantigen. Immunoliposomes specific to DC can be used targeted toa DC-specific surface molecule such as DEC-205, CD11c or CD83, the siRNAmay be administered systemically in vivo, in a manner to target DC inhomeostatic conditions.

To summarize, the present invention provides novel transformed immunecells which exhibit a targeted gene-specific knockout phenotype in orderthat such cells can be used therapeutically to modulate immune responsesin a mammal via alteration of T cell activity. The present inventionprovides novel altered DC that do not express one or more genes encodinga surface marker, chemokine, cytokine, enzyme or transcriptional factorthat are involved in DC activity, and as such, suppress or stimulateimmune system functioning via the modulation of T cell activity.

The present invention also encompasses therapeutic methods for thetreatment of a variety of immune disorders with the use of the alteredimmune cells or with the use of the siRNA. In embodiments of theinvention, the immune cells is a DC that is transfected in vitro toproduce a desired DC phenotype and then used ex vivo as a perfusioncomposition for a transplantation tissue or organ or in vivo asadministered to a mammalian subject. The invention also encompasses thein vivo use of siRNA directed to selected molecules associated withimmune cells in order to alter T cell activity and thus treat a varietyof immune disorders.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLES Example 1 Generation of Bone Marrow-Derived DC

DC were generated from bone marrow progenitor cells as previouslydescribed (22). Briefly, bone marrow cells were flushed from the femursand tibias of C57BL/6 mice (Jackson Labs, Bar Harbor Me.), washed andcultured in 24-well plates (2×10⁶ cells/ml) in 2 ml of complete medium(RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/ml of penicillin,100 μg of streptomycin, 50 μM 2-mercaptoethanol, and 10% fetal calfserum (all from Life Technologies, Ontario, Canada) supplemented withrecombinant GM-CSF (10 ng/ml; Peprotech, Rocky Hill, N.J.) andrecombinant mouse IL-4 (10 ng/ml; Peprotech). All cultures wereincubated at 37° C. in 5% humidified CO₂. Non-adherent granulocytes wereremoved after 48 hrs of culture and fresh medium was added. After 7 daysof culture >90% of the cells expressed characteristic DC specificmarkers as determined by FACS. DC were washed and plated in 24-wellplates at a concentration of 2×10⁵ cells per well in 400 μl ofserum-free RPMI-1640.

Example 2 siRNA Synthesis and Transfection

The siRNA sequences were selected according to the method of Elbashir etal (23). The siRNA sequences specific for IL-12p35(AACCUGCUGMGGAUGGUGAC), IL-12p40 (MGAUG ACAUCACCUGGACCU), and IFN-γ(MCTGGCAAAAGGATGGTGAC) were synthesized and annealed by the manufacturer(Dharmacon Inc. Lafayette, Co.). siRNA for IFN-γ was used as a controlsince bone marrow derived DC generated by the conditions described abovedid not produce IFN-γ after stimulation. Transfection efficiencies weredetermined using unlabeled and fluorescein labeled siRNA Luciferase GL2Duplex (Dharmacon Inc). Transfection was carried out as describedpreviously (Elbashir, S. M., 2002. Methods 26:199). Briefly, 3 μl of 20μM annealed siRNA was incubated with 3 μl of GenePorter (Gene TherapySystems, San Diego, Calif.) in a volume of 100 μl RPMI-1640 (serum free)at room temperature for 30 min. This was then added to 400 μl of DC cellculture as described above. Mock controls were transfected with 3 μlGenePorter alone. After 4 hrs of incubation an equal volume of RPMI-1640supplemented with 20% FCS was added to the cells. 24-48 hrs later,transfected DC were washed and used for subsequent experiments.

In the transfection by phagocytosis, bone marrow DC progenitors at day 4of culture were incubated in a final concentration of 60 pMFL-siRNA-Luc. Cells remained in culture with GM-CSF and IL-4 asdescribed above. At day 8 of culture cells were activated with LPS/TNF-αand incorporated FL-siRNA-Luc was assessed by flow cytometry on day 9.

Example 3 DC Activation and MLR

Transfected DC (1×10⁶ cells) were plated in 24 well plates andstimulated with LPS (10 ng/ml, Sigma Aldrich, St Louis, Mo.)+TNFα (10ng/ml, Peprotech) for 48 hrs, at which point supematants were used forELISA and RNA was extracted from the cells for RT-PCR. For mixedleukocyte reaction (MLR), T cells were purified from BALB/c splenocytesusing nylon wool columns and were used as responders (1×10^(6/)well).siRNA-treated DC (5-40×10³, from C57/BL6 mice) were used as stimulators.72 hour MLR was performed and the cells were pulsed with 1 μCi[³H]-thymidine for the last 18 hrs. The cultures were harvested on toglass fiber filters (Wallac, Turku, Finland). Radioactivity was countedusing a Wallac 1450 Microbeta liquid scintillation counter and the datawere analyzed with UltraTerm 3 software.

Example 4 Flow Cytometry

Phenotypic analysis of siRNA-treated DC was performed on a FACScan(Becton Dickinson, San Jose, Calif.) and analyzed using CellQuestsoftware (Becton Dickinson). The following FITC conjugated anti-mousemAbs were used: anti-I-A^(b), anti-CD11c, anti-CD40, and anti-CD86 (BDPharMingen, San Diego, Calif.). The annexin-V/propidium iodide method ofdetermining apoptosis/necrosis was used as previously described (Min W.P., 2000. J Immunol 164:161). All flow cytometric analyses wereperformed using appropriate isotype controls (Cedariane Laboratories,Homby ON, Canada).

Examble 5 RT-PCR

Total RNA from siRNA-treated DC (10⁶ cells) or from T cells purifiedfrom MLR (10⁶ cells) was isolated by TRIzol reagent (Gibco BRL)according to the manufacturer's instructions. First strand CDNA wassynthesized using an RNA PCR kit (Gibco BRL) with the supplied oligod(T)16 primer. One μmol of reverse transcription reaction product wasused for the subsequent PCR reaction. The primers used for IL-12p35 andIL-12p40 flanked the sequences targeted by siRNA (IL-12p35, forwardprimer 5′-GCCAGGTGTCTTAGCCAGTC-3′, reverse primer5′-GCTCCCTCTTGTTGTGGMG-3′; IL-12p40, forward primer5′-ATCGTTTTGCTGGTGTCTCC-3′, reverse primer 5′- CTTTGTGGCAGGTGTACTGG-3′).In addition, IL-10, IFN-γ, IL-4 and GAPDH (internal control) primerswere used as previously described (Zhu, X., et. al., 1994.Transplantation 58:1104). The PCR conditions were: 94° C. for 1 min, 60°C. for 1 min, and 72° C. for 1 min, and PCR was done for 35 cycles. PCRproducts were visualized with ethidium bromide on 1.5% agarose gel.

Example 6 Enzyme-Linked Immunosorbent Assay (ELISA)

The siRNA-treated DC (10⁵, C57/BL6 origin) were cultured with theallogeneic T cells (1×10⁶) for 48 hrs. The supernatants were harvestedand assessed for DC cytokines (IL-12p70, IL-10) and T cell cytokines(IFN-γ, IL-4) by ELISA. Cytokine specific ELISA (Endogen, Rockford,Ill.) was used for detecting cytokine concentrations in culturesupematants according to the manufacturer's instructions using aBenchmark Microplate Reader (Bio-Rad Laboratories).

Example 7 Immunization of Mice with Peptide-Pulsed DC

Day 7 bone marrow-derived DC were transfection with siRNA-IL12p35, ortransfection reagent alone as described above, and pulsed with 10 μg/mlof keyhole limpet hemocyanin (KLH) (Sigma-Aldrich Rockford Ill.) for 24hrs. DC were then activated with LPS+TNFα for 24hrs, washed extensivelyand used for subsequent transfer experiments. Antigen-pulsed DC (5×10⁵cells/mouse) were injected subcutaneously into syngeneic mice. Mice weresacrificed after 10 days and cell suspensions were prepared from thedraining lymph nodes. These cells were cultured in 96-well plates at aconcentration of 4×10⁵ cells/well in the presence or absence of antigenfor 48 hrs at which point culture supernatants were used for analysingcytokine production by ELISA.

For statistical analysis, one-way ANOVA followed by the Newman KeulsTest was used to determine the significance between groups for cytokineproduction and MLR. Differences with p-values less than 0.05 wereconsidered significant.

Although preferred embodiments have been described herein in detail itis understood by those of skill in the art that using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein can be made. Such equivalents areintended to be encompassed by the scope of the claims appended hereto.

1. A mammalian immune cell exhibiting a targeted endogenousgene-specific knockout phenotype, said immune cell altering an immuneresponse in a mammal via the modulation of T cell activity.
 2. Theimmune cell of claim 1, wherein said cell comprises a construct thatinhibits the expression of said endogenous target gene.
 3. The immunecell of claim 2, wherein said construct is selected from the groupconsisting of siRNA and hybrid DNA/RNA/
 4. The immune cell of claim 1,wherein said endogenous gene encodes a surface marker, a chemokine, acytokine, an enzyme or a transcriptional factor.
 5. The immune cell ofclaim 1, wherein said immune cell is selected from the group consistingof an endothelial cell and an antigen presenting cell.
 6. The immunecell of claim 5, wherein said antigen presenting cell is selected fromthe group consisting of a dendritic cell, a macrophage, a myeloid cell,a B lymphocyte and mixtures thereof.
 7. The immune cell of claim 6,wherein said immune cell is a dendritic cell.
 8. The immune cell ofclaim 7, wherein said dendritic cell is activated.
 9. The immune cell ofof claim 1, wherein said siRNA or hybrid DNA/RNA is provided within aplasmid or vector.
 10. The immune cell of claim 9, wherein said plasmidor vector additionally comprises an expressible nucleic acid sequenceencoding an antigen.
 11. The immune cell of claim 7 or 8, wherein saiddendritic cell additionally comprises tumor cell mRNA.
 12. The immunecell of claim 4, wherein said surface marker, chemokine, cytokine,enzyme or transcription factor is selected from the group consisting ofTNFα, IL-1, IL-1b, IL-2, TNFβ, IL-6, IL-7, IL-8, IL-23, IL-15, IL-18,IL-12, IFNγ, IFNα, lymphotoxin, DEC-25, CD11c, CD40, CD80, CD86, MHCI,MHCII, ICAM-1, TRANCE, CD200, CD200 receptor, CD83, CD2, CD44, CD91,TLR-4, TLR-9, 4-1BBL, nicotinic receptor, GITR-L, OX-40L, CD-CK1,TARC/CCL17, CCL3, CCL4, CXCL9, CXCL10, IKK-β, NK-κB, STAT4, ICSBP/IFN,regulatory factor 8, TRAIL, Inos, arginase, FcgammaRI and II, thrombin,MIP-1α and MIP-1B.
 13. The immune cell of claim 12, wherein saidcytokine is selected from IL-12 and TNFα.
 14. The immune cell of claim12, wherein said immune cell inhibits T cell activity.
 15. The immunecell of claim 4, wherein said surface marker and enzyme are selectedfrom the group consisting of B7-H1, EP2, IL-10 receptor, VEGF-receptor,CD101, PD-L1, PD-L2, HLA-11, DEC-205, CD36 and indoleamone2,3-dioxygenase.
 16. The immune cell of claim 15, wherein said immunecell stimulates T cell activity.
 17. The immune cell of claim 14 or 16,wherein said immune cell is administered to a mammalian subject for thetreatment of an immune disorder.
 18. The immune cell of claim 17,wherein said immune disorder is selected from the group consisting ofseptic shock, rheumatoid arthritis, transplant rejection, scleroderma,immune medicated diabetes, chronic inflammatory bowel syndrome, HIV,cancer, colitis, Crohn's disease, Goodpasture's syndrome, MultipleSclerosis, Grave's disease, Hashimoto's thyroditis, Autoimmunepernicious anemia, Autoimmune Addison's disease, Vitiligo, Myastheniagravis, Scleroderma, Systemic lupus erythematosus, Primary Sjogren'ssyndrome, Polymyositis, Pemphigus vulgaris, Ankylosing spondylitis,Acute anterior uveitis, Hypoglycemia and inflammation associated withchronic illness.
 19. The immune cell of claim 1, wherein said immunecell is provided as a composition comprising a pharmaceuticallyacceptable carrier.
 20. The immune cell of claim 19, wherein saidcomposition additionally comprises an adjuvant and/or antigen.
 21. Amedicament for the treatment of an immune disorder characterized byinappropriate T cell activity, said medicament comprising a mammalianimmune cell that exhibits a targeted gene-specific knockout phenotype,wherein said gene is selected from one or more of a surface marker, achemokine, a cytokine, an enzyme and a transcriptional factor.
 22. Amedicament for the treatment of an immune disorder characterized byinappropriate T cell activity, said medicament comprising a siRNApossessing specific homology to part or the entire exon region of a geneencoding a surface marker, a chemokine, a cytokine, an enzyme or atranscriptional factor or an antigen presenting cell (APC).
 23. Themedicament of claim 20 or 21, wherein said gene is selected from thegroup consisting of TNFα, IL-1, IL-1b, IL-2, TNFβ, IL-6, IL-7, IL-8,IL-23, IL-15, IL-18, IL-12, IFNα, lymphotoxin, DEC-25, CD11c, CD40,CD80, CD86, MHCI, MHCII, ICAM-1, TRANCE, CD200, CD200 receptor, CD83,CD2, CD44, CD91, TLR4, TLR-9, 4-1BBL, nicotinic receptor, GITR-L, OX40L,CD-CK1, TARC/CCL17, CCL3, CCL4, CXCL9, CXCL10, IKK-β, NF-κB, STAT4,ICSBP/IFN, regulatory factor 8, TRAIL, Inos, arginase, FcgammaRI and II,thrombin, MIP-1α and MIP-1B.
 24. The medicament of claim 22, whereinsaid T cell activity is inhibited.
 25. The medicament of claim 20 or 21,wherein said gene is selected from the group consisting of B7-H1, EP2,IL-10 receptor, VEGF-receptor, CD101, PD-L1, PD-L2, HLA-11, DEC-205,CD36 and indoleamine 2,3-dioxygenase.
 26. The medicament of claim 25,wherein said T cell activity is stimulated.
 27. The medicament of claim23 or 24, wherein said immune disorder is selected from the groupconsisting of septic shock, rheumatoid arthritis, transplant rejection,scleroderma, immune mediated diabetes, chronic inflammatory bowelsyndrome, HIV, cancer, colitis, Crohn's disease, Goodpasture's syndrome,Multiple Sclerosis, Grave's disease, Hashimoto's thyroditis, Autoimmunepernicious anemia, Autoimmune Addison's disease, Vitiligo, Myastheniagravis, Scleroderma, Systemic lupus erythermaosus, Primary Sjogren'ssyndrome, Polymyositis, Pemphigus vulgaris, Ankylosing spondylitis,Acute anterior uveitis, Hypoglycemia and inflammation associated withchronic illness.
 28. The medicament of claims 21 to 22 wherein saidimmune cell is selected from an endothelial cell and an antigenpresenting cell (APC).
 29. The medicament of claim 28, wherein saidantigen presenting cell is selected from the group consisting of adendritic cell, a macrophage, a myeloid cell, a B lymphocyte andmixtures thereof.
 30. The medicament of claim 29, wherein said immunecell is a dendritic cell.
 31. The medicament of claim 30, wherein saiddendritic cell is activated.
 32. A composition for the treatment of animmune disorder, said composition comprising at least one of: (a) aconstruct that inhibits the expression of an endogenous target geneencoding a surface marker, a chemokine, a cytokine, an enzyme or atranscriptional factor in an immune cell such that said immune cellsalters T cell activity; and (b) an immune cell wherein said immune cellcomprises at least one construct that inhibits the expression of anendogenous target gene encoding a surface marker, a chemokine, acytokine, an enzyme or a transcriptional factor; and (c) apharmaceutically acceptable carrier, wherein said composition alters Tcell activity leading to an altered immune response.
 33. The compositionof claim 32, wherein said construct is selected from the groupconsisting of siRNA and hybrid DNA/RNA.
 34. The composition of claim 32,wherein said immune cell is selected from the group consisting of anendothelial cell and an antigen presenting cell.
 35. The composition ofclaim 34, wherein said antigen presenting cell is selected from thegroup consisting of a dendritic cell, a macrophage, a myeloid cell, a Blymphocyte and mixtures thereof.
 36. The composition of claim 35,wherein said immune cell is a dendritic cell.
 37. The composition ofclaim 36, wherein said dendritic cell is activated.
 38. The compositionof claim 33, wherein said siRNA or hybrid DNA/RNA is provided within aplasmid or vector.
 39. The composition of claim 38, wherein said plasmidor vector additionally comprises an expressible nucleic acid sequenceencoding an antigen.
 40. The composition of claim 35, wherein saiddendritic cell additionally comprises tumor cell mRNA.
 41. Thecomposition of claim 32, wherein said surface marker, chemokine,cytokine, enzyme or transcription factor is selected from the groupconsisting of TNFα, IL-1, IL-1b, IL-2, TNFβ, IL-6, IL-7, IL-8, IL-23,IL-15, IL18, IL-12, IFNγ, IFNα, lymphotoxin, DEC-25, CD11c, CD40, CD80,CD86, MHCI, MHCII, ICAM-1, TRANCE, CD200, CD200 receptor, CD83, CD2,CD44, CD91, TLR4, TLR-9, 4-1BBL, nicotinic receptor, GITR-L, OX-40L,CD-CK1, TARC/CCL17, CCL3, CCL4, CXCL9, CXCL10, IKK-β, NF-κB, STAT4,ICSBP/IFN, regulatory factor 8, TRAIL, Inos, arginase, FcgammaRI and II,thrombin, MIP-1α and MIP-1B.
 42. The composition of claim 41, whereinsaid cytokine is selected from IL-12 and TNFα.
 43. The composition ofclaim 32, wherein said surface marker and enzyme are selected from thegroup consisting of B7-H1, EP2, IL-10 receptor, VEGF-receptor, CD101,PD-L1, PD-L2, HLA-11, DEC-205, CD36 and indoleamine 2,3-dioxygenase. 44.The composition of claim 32, wherein said immune disorder is selectedfrom the group consisting of septic shock, rheumatoid arthritis,transplant rejection, scleroderma, immune mediated diabetes, chronicinflammatory bowel syndrome, HIV, cancer, colitis, Crohn's disease,Goodpasture's syndrome, Multiple Sclerosis, Grave's disease, Hashimoto'sthyroditis, Autoimmune pernicious anemia, Autoimmune Addison's disease,Vitiligo, Myasthenia gravis, Scleroderma, Systemic lupus erythermaosus,Primary Sjogren's syndrome, Polymyositis, Pemphigus vulgaris, Ankylosingspondylitis, Acute anterior uveitis, Hypoglycemia and inflammationassociated with chronic illness.
 45. The composition of claim 32,wherein said composition is used to perfuse tissues and/or organs exvivo.
 46. A method for inhibiting the T cell activating ability of a DC,the method comprising transforming said DC with a constructcapable ofinhibiting the expression of an endogenous target gene encoding asurface marker, a chemokine, a cytokine, an enzyme or a transcriptionalfactor.
 47. A method of decreasing the immunogenicity and rejectionpotential of an organ for transplantation, said method comprisingperfusing said organ with a composition that suppresses T cell activity,said composition comprising at least one construct that inhibits theexpression of an endogenous target gene encoding a surface marker, achemokine, a cytokine, an enzyme or a transcriptional factor and apharmaceutically acceptable carrier.
 48. The method of claim 46 or 47,wherein said construct is selected from siRNA and hybrid DNA/RNA. 49.The method of claim 48, wherein said siRNA is provided within an antigenpresenting immune cell.
 50. A method for making a immune cell thatalters that activity of T cell in vivo, said method comprising;transforming immune cells in vitro with at least one construct thatinhibits the expression of an endogenous target gene encoding a surfacemarker, a chemokine, a cytokine, an enzyme or a transcriptional factor.51. A method for the treatment of autoimmune disorders andtransplantation rejection in a mammalian subject, said method comprisingadministering a therapeutically effective amount of a composition tosaid subject, said composition comprising DC that contain at least oneconstruct that inhibits the expression of an endogenous target geneencoding a surface marker, a chemokine, a cytokine, an enzyme oratranscriptional factor, wherein said DC suppresses T cell activity. 52.The method of claim 50 or 51, wherein said construct is selected fromsiRNA and hybridDNA/RNA.
 53. A method for the treatment of autoimmunedisorders and transplantation rejection in a mammalian subject, saidmethod comprising administering a therapeutical effective amount of acomposition to said subject, said composition comprising an siRNAtargeted to inhibit expression of an endogenous target gene in anantigen presenting cell, said gene encoding a surface marker, achemokine, a cytokine, an enzyme or a transcriptional factor, whereinsaid siRNA suppresses T cell activity.
 54. The method of claims 51, 52or 53, wherein said autoimmune disorder is selected from the groupconsisting of septic shock, rheumatoid arthritis, transplant rejection,scleroderma, immune mediated diabetes, chronic inflammatory bowelsyndrome, HIV, cancer, colitis, Crohn's disease, Goodpasture's syndrome,Multiple Sclerosis, Grave's disease, Hashimoto's thyroditis, Autoimmunepernicious anemia, Autoimmune Addison's disease, Vitiligo, Myastheniagravis, Scleroderma, Systemic lupus erythematosus, Primary Sjogren'ssyndrome, Polymyositis, Pemphigus vulgaris, Ankylosing spondylitis,Acute anterior uveitis, Hypoglycemia and inflammation associated withchronic illness.